Abstract: A major first step in audition is the conversion of mechanical deflection of the sensory hair bundle into an electrical signal that drives the hair cell receptor potential that in turn controls cochlear amplification (outer hair cells) and synaptic transmission (inner hair cells). Alterations in any step of this process will degrade signal processing throughout the auditory pathway. During the previous funding period, we demonstrated that mammalian cochlea hair bundles do not move coherently, meaning that there were both temporal and magnitude differences between stereocilia motion depending on the mode of stimulation. The lack of coherence leads to very different mechanotransducer current responses, which are predicted to generate very different receptor potentials. This finding drives a major component of the present proposal which is focused upon identifying how hair bundles move in situ, to better understand the properties of the physiologically driven mechanotransducer currents. We have developed new technology including imaging bundle movement in situ at high speeds as well as image processing algorithms that can detect motions at below 2 nm. We will use these new technologies as well as high speed calcium imaging, new fluid jet and stiff probe stimulating devices and electrophysiological tools to investigate stereocilia motion. Results from these experiments will shed light onto why outer hair cells are embedded in the tectorial membrane and inner hair cells are not. We will also identify the hair bundle and tectorial membrane mechanical properties responsible for regulating hair bundle motion. During the past funding period we also identified several mechanisms by which the lipid membrane either directly or indirectly alters mechanotransduction currents. We have developed new technologies including fluorescence recovery after photobleaching to directly assess the mechanical properties of the lipid bilayer as a means of identifying the underlying mechanisms for regulating the mechanotransducer channel. We will use these new technologies coupled with pharmacological manipulations of the lipid bilayer to investigate the role of specific lipids and their mechanical properties in modulating hair cell MET.